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In a negative control for PCR experiment, a reaction mixture added with pure water instead of DNA extract sometimes generated a slightly visible PCR band of 16S rRNA gene fragments. The retrieved sequences always indicated 99% identity to that of Nesterenkonia aethiopica in the class Actinobacteria. We considered enrichment cultures from which sequences similar to the negative control sequence were detected as growth-negative.

Anaerobic enrichments from rock cores

Cultivations targeting anaerobic hydrogenotrophic methanogens were performed at three different temperatures of 15°C, 37°C, and 55°C, and each headspace gas was examined by gas chromatography analysis; however, methane production was not detected from any of the enrichment cultures. The other targeted anaerobic metabolism was hydrogen oxidation. The medium containing hydrogen as an electron donor and sulfate and nitrate as electron acceptors was used for enrichments. Apparent microbial growth was not detected in any of the enrichment cultures at 15°C, whereas the enrichment cultures at 37°C showed bacterial growth from nine core samples (Table T2). The detected bacteria were affiliated to the genera Bacillus, Methylobacterium, and Sphingomonas by direct sequencing analysis of 16S rRNA genes. Bacillus spp. have previously been cultivated from subsurface basaltic cores and the inside of seafloor basaltic rocks (Lysnes et al., 2004; Rathsack et al., 2009). Although two Bacillus spp., Bacillus schlegelii and Bacillus tusciae, were previously known to be facultatively chemolithoautotrophic hydrogen-oxidizing bacteria, these species have recently been reclassified in novel genera Hydrogenibacillus (Kämpfer et al., 2013) and Kyrpidia (Klenk et al., 2011), respectively. To our knowledge, no other Bacillus spp. are reported to be capable of hydrogen oxidation.

The archaeal 16S rRNA gene was not amplified from any of the anaerobic cultures.

Aerobic enrichments from rock cores

For aerobic cultivations, reduced sulfur compounds (elemental sulfur and thiosulfate) and hydrogen were used as electron donors. Two enrichment cultures targeting sulfur oxidizers indicated bacterial growth, and Moraxella sp. was detected from one of the two examined cultures. Enrichments targeting hydrogen oxidizers were more prolific than those targeting sulfur oxidizers. Bacterial growth was detected from 10, 8, and 13 core samples cultivated at 8°C, 25°C, and 37°C, respectively (Table T3).

Members of the genera Ralstonia and Pseudomonas were most frequently detected in the enrichment cultures at 8°C and 25°C. Ralstonia and Pseudomonas spp. have previously been detected in cultivation analyses of the subsurface and seafloor basaltic rocks and also in culture-independent microbial community analyses of deep subsurface gabbroic rock cores (Lysnes et al., 2004; Mason et al., 2010; Rathsack et al., 2009). Ralstonia spp. are known to be tough microorganisms (Mijnendonckx et al., 2013), and they might be living in various harsh environments, including nutrient-starved endolithic habitats. In the genus Ralstonia, only one species, Ralstonia eutropha, was previously known to be a facultatively chemolithoautotrophic hydrogen-oxidizing bacterium; however, R. eutropha was reclassified in the genus Cupriavidus later and now is recognized as a synonym of Cupriavidus necator (Vandamme and Coenye, 2004). Currently, it seems that there is no published data about chemolithotrophic hydrogen-oxidizing species of the genera Ralstonia and Pseudomonas.

A PCR amplification of the culture of the sample from Section 336-U1382A-6R-1A at 8°C generated a dense product band, but direct sequencing of the product did not succeed. The product was then cloned, and 16S rRNA gene sequences similar to those of Salinibacterium and Sphingomonas spp. were obtained. Salinibacterium amurskyense is a marine heterotroph capable of growing at 4°C (Han et al., 2003). The genus Sphingomonas is known for the ability to degrade a wide range of recalcitrant environmental pollutants (Yabuuchi and Kosako, 2005). In the enrichment culture of Section 336-U1383C-24R-1A at 25°C, visible growth of black fungi-like cells was observed, and then the culture was analyzed separately as described below.

In the enrichment cultures at 37°C, Paenibacillus and Acidovorax spp. were frequently detected. A Paenibacillus species has previously been cultivated from subsurface basaltic cores (Lysnes et al., 2004). Several Paenibacillus spp. have been associated with Fe(III) reduction and have been reported to predominate in the Fe(III)-reducing consortia of subsurface sediments in terrestrial heavy metal–contaminated sites (Ahmed et al., 2012; Petrie et al., 2003). Although the genus Acidovorax is generally characterized by chemoorganotrophic growth, Acidovorax ebreus has reported as a mixotroph to utilize Fe(II) as the electron donor (Byrne-Bailey et al., 2010). The lithoautotrophic growth by hydrogen oxidation has also been reported in strains of two species of the genus Acidovorax, Acidovorax facilis and Acidovorax delafieldii (Willems et al., 1990).

The archaeal 16S rRNA gene was not amplified from any of the aerobic cultures.

Detection of the mcrA gene of methanogens from sediment cores

To select sediment cores suitable for the cultivation of methanogens, amplification of the mcrA genes in DNA from sediment cores was attempted using real-time PCR. The mcrA gene was, however, not detected in any of the sediment cores (Table T2). Therefore, cultivation of methanogens was not performed on the sediment cores.

Enrichments from sediment cores

Sediment core samples were used for inoculation of aerobic enrichments in the medium for sulfur oxidizers at temperatures of 15°C and 37°C. Bacterial growth was observed in seven core samples at 15°C, whereas there was no clear indication of microbial growth in the enrichment cultures at 37°C (Table T4). The bacteria detected in the cultures at 15°C were members of the genera Pseudomonas, Halomonas, Marinobacter, and Paracoccus. Members of these genera are known to be typical inhabitants of subsurface sediments (Parkes et al., 2014). It has been reported that strains belonging to these genera are obligately heterotrophic sulfur oxidizers, which means they can utilize sulfur compounds as electron donors and organic compounds as carbon sources, or facultatively autotrophic sulfur oxidizers (Petri et al., 2001; Sorokin, 2003; Van Spanning, 2005). In thiosulfate-oxidizing heterotrophic Pseudomonas stutzeri, the presence of thiosulfate dehydrogenase participating in thiosulfate oxidation and the exhibition of it’s enzymatic activity were proven by using an expressed recombinant protein (Denkmann et al., 2012).

The archaeal 16S rRNA gene was not amplified from any of the aerobic cultures.

A fungal isolate from the rock core

Fungal growth was observed in the enriched culture from Core 336-U1383C-24R-1A rock at 25°C as described above. The isolated fungal strain was designated NPf1. A sequence of ~4.4 kbp including 18S, 5.8S, and 28S rRNA coding regions and the internal transcribed spacer regions (ITS1 and ITS2) was obtained from the isolate (GenBank/EMBL/DDBJ accession number LC017736). The isolate NPf1 was affiliated to the genus Exophiala of the order Chaetothyriales by similarity analysis (Fig. F1). It has been reported that many fungal isolates from terrestrial rocks are grouped into the order Chaetothyriales (Sterflinger et al., 1997; Ruibal et al., 2008).

We succeeded in retrieving partial 16S rRNA gene sequences by the direct sequencing of PCR products from most of the growth-positive enrichment cultures, suggesting that the detected microorganisms were certainly grown in the enrichment cultures. However, we did not achieve the subcultivation of the detected bacteria in the respective original media in any case. Because the enrichment cultures contained rock particles as inocula transferred from the slurry, rock minerals might be needed for their growth.